This project seeks to analyze the mechanism by which dual-specificity tyrosine-phosphorylation-regulated protein kinases (DYRKs) are activated. Protein phosphorylation by kinases is crucial for cell signaling pathways. Abnormal phosphorylation contributes to a large number of human diseases and hence protein kinases represent a point for therapeutic intervention. The DYRK family of kinases for example, plays important roles in Down Syndrome, microcephaly, cancer, and Alzheimer's disease. One of the most important modes whereby protein kinases are activated is by the phosphorylation of key residues in the activation loop of the kinase domain. DYRKs and glycogen synthase kinase 3 (GSK3) enzymes are dual-specificity kinases that autophosphorylate their activation loop on an essential tyrosine but phosphorylate their substrates on serine and threonine. Work in this lab has shown that autophosphorylation of the critical activation-loop tyrosine is intra-molecular and mediated by a transitory intermediate form of the kinase during the initial folding of the molecule. The intermediates differ in residue and substrate specificity, and sensitivity to small-molecule inhibitors, compared to their mature counterpart. The intermediate form of the kinase therefore represents a previously unrecognized novel target for drug inhibition. The transitional-intermediate form of GSK3 requires the chaperone activity of hsp90. Preliminary studies in this lab, indicates that class 2 DYRKs also require the activity of a highly conserved intra-molecular chaperone-like region (the NAPA-region) for autophosphorylation. This project is to further analyze the function of the NAPA- region. It is likely that the highly conserved nature of the sequence will allow the design of small molecule activators and inhibitors of the DYRKs that are likely to be of therapeutic use. The specific aims of this project are to (1) determine the role of the NAPA-region in class 2 DYRK autophosphorylation, (2) characterize the ability of isolated class 2 N-termini to complement autophosphorylation in trans, (3) initiate studies to characterize mechanism of class 1 DYRK tyrosine autophosphorylation focusing on human DYRK1A and Drosophila MNB/DYRK1A, and (4) conduct DYRK inhibitor studies. Our in vivo assays will form the basis for genetic screens to identify the role of DYRKs in developmentally important signaling pathways, and will provide the basis for functional screens of small molecule inhibitors. Long term goals of this lab are to identify the surface region in the kinase domain that interacts with the NAPA- region, to establish if a comparable region is present in GSK3 and to determine if this chaperone-mediated event is a wide-spread mechanism of protein kinase activation. This knowledge will form the basis for the rational design of inhibitors and activators of DYRK, GSK3 and possibly other protein kinases.